ABSTRACT Autism spectrum disorders (ASD) are neurodevelopmental disorders with a prevalence of 1:68 in children. Although several genetic factors have been implicated in ASD etiology, the exact cause of ASD is unknown, and no treatment directly targets specific pathways in the brain. We hypothesize that peptides and proteins involved in the pathogenesis of ASD demonstrate regional expression patterns in brains with ASD which differ from those expressed in normal brains. Such molecules can be potential biomarkers for early identification of individuals at risk for developing ASD, and some may even constitute novel therapeutic targets. Experimental findings that metabotropic glutamate receptor 5 (mGluR5) antagonists reverse autism phenotypes in several mouse models of autism support this hypothesis in that a single targeted pharmacological intervention alleviates autism symptoms regardless of underlying etiology. In order to test and evaluate this hypothesis, a technology enabling simultaneous characterization, visualization and quantification of proteins and peptides directly from brain tissue is urgently needed. Herein we propose to develop and expand the analytical capability of the current state-of- the-art mass spectrometry imaging (MSI) technology to address this scientific need. We identify several remaining challenges facing MALDI MSI, including: (1) Difficulty in protein identification directly from tissue without homogenization and liquid chromatography (LC) tandem mass analysis; (2) Difficulty in producing efficient fragmentation from singly charged ions inherent with the MALDI ionization process; (3) Limited m/z range of high resolution accurate mass (HRAM) MS platforms for large protein in situ characterization; and (4) Difficulty in accurate quantification of proteins and peptides directly from complex tissue samples. To overcome these critical challenges, we propose the development of next generation of MALDI MSI platform and the application of this novel platform to autism research. We will combine recently developed multiply charged laserspray ionization (LSI) and matrix assisted ionization in vacuum (MAIV) techniques with in situ enzymatic digestion and novel multiplex MSI strategies using HRAM mass spectrometers, for enhanced analytical performance and proteome coverage. Furthermore, novel in situ isotopic chemical labeling and antibody based tag-mass strategies will be developed to enable quantification of biomolecules in brain sections from control and ASD mouse models to validate potential biomarkers. Such molecular validation will be further supported by pharmacological treatment and behavioral testing. The proposed research will create improved MALDI MSI technology that enables simultaneous mapping and quantifying in situ protein expression patterns in biological tissues from animal models of diseases. The proposed research has tremendous translational relevance, as successful completion would reveal potential early diagnostic biomarkers for ASD, which could be detected in subjects at risk by means of magnetic resonance spectroscopy, positron emission tomography (PET) or biochemical methods. These same biomarkers could also represent potential novel therapeutic targets.